Eur Respir J 2011; 37: 1522–1533 DOI: 10.1183/09031936.00172510 CopyrightßERS 2011
LETTERS
Macrolide-resistant Mycoplasma pneumoniae in paediatric pneumonia To the Editors: Mycoplasma pneumoniae is one of the most common causes of bacterial community-acquired pneumonia (CAP) in paediatrics, and can lead to severe and long-lasting disease [1]. Macrolides are usually considered the first-choice antimicrobials for M. pneumoniae CAP in children because the alternatives (i.e. fluoroquinolones and tetracyclines) are not approved for use in the first years of life [2]. Recent studies from Japan and China have shown macrolide resistance in up to 80% of M. pneumoniae strains [3, 4], but it has been detected in relatively few cases in the USA, France and Germany, and not at all in other European countries [5–7]. The mechanism of M. pneumoniae macrolide resistance is related to point mutations in domain V of the 23S rRNA gene of M. pneumoniae and macrolide resistance is usually detected at disease onset [4]. We here describe the first case of macrolide-resistant M. pneumoniae detected during treatment with clarithromycin in an otherwise healthy child with CAP. An otherwise healthy 6-yr-old girl with an unremarkable medical history who had never travelled abroad was admitted in Bari, Italy, after suffering from a dry cough for 3 days with fever up to 40.5uC, accompanied by increasing malaise and dyspnoea. Upon admission, she was severely ill, with a high temperature, lethargy, an increased respiratory rate (60 breaths?min-1), tachycardia and normal blood pressure. Room air oximetry revealed 85% oxygen saturation, whereas arterial gas sampling showed severe hypoxaemia (50 mmHg) with hypocapnia and a normal pH. Other routine blood examinations revealed neutrophilia with increased C-reactive protein levels and a high erythrocyte sedimentation rate. A physical examination revealed diffuse crackles with reduced vesicular sounds on both lungs, and chest radiography showed an interstitial pattern with multiple ‘‘ground-glass’’ infiltrates. Blood, nasopharyngeal swab and sputum samples were immediately obtained for serology, real-time PCR, and respiratory virus and bacterial cultures. High-flow oxygen (8 L?min-1) was necessary to maintain saturation above 90%, and the girl also received nebulised albuterol, intravenous antibiotics (ampicillin/sulbactam 150 mg?kg-1?day-1 i.v. in three doses and clarithromycin 7.5 mg?kg-1?day-1 i.v. in two doses) and cristalloids. Table 1 shows the clinical and laboratory findings upon admission and during the course of hospitalisation. The results of microbiological tests of the samples obtained at admission (available over the following 3 days) were negative for respiratory syncytial virus, adenovirus, influenza viruses, parainfluenza viruses, human metapneumovirus, human bocavirus, coronaviruses, Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Staphylococcus aureus, Mycobacterium 1522
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tuberculosis, Chlamydophila pneumoniae and Legionella pneumophila. However, serology and nasopharyngeal swab real-time PCR (performed at day 0 and available after 3 days) were positive for acute M. pneumoniae infection (specific immunoglobulin (Ig)M 1:300 and specific IgG 1:800; real-time PCR positive for M. pneumoniae DNA). Because of the persistence of severe hypoxaemia and the girl’s poor general condition, she was transferred to an intensive care unit (ICU) for monitoring after 4 days. A chest computed tomography (CT) scan performed in the ICU showed further progression of the lung infiltration with multiple bilateral areas of consolidation in both lungs and diffuse interstitial infiltration. The same supportive therapy was continued with the administration of clarithromycin (7.5 mg?kg-1?day-1 i.v. in two doses), meropenem (100 mg?kg-1?day-1 i.v. in three doses) and teicoplanin (10 mg?kg-1?day-1 i.v. o.d.) instead of ampicillin/ sulbactam. A broad-spectrum antibiotic therapy was continued despite the detection of only M. pneumoniae because of the severity of the disease. Further microbiological tests of blood, nasopharyngeal swab and sputum samples continued to show M. pneumoniae DNA on the nasopharyngeal swab and no other pathogen. Despite the broad-spectrum antibiotic therapy, the child remained severely ill and, on day 10, nasopharyngeal aspiration was repeated and a bronchoalveolar lavage sample was obtained: real-time PCR revealed M. pneumoniae DNA in both samples. Given the failure of 10 days of i.v. clarithromycin therapy and the persistence of a positive M. pneumoniae PCR, a macrolideresistant M. pneumoniae infection was hypothesised and clarithromycin was replaced by ciprofloxacin (40 mg?kg-1?day-1 in two doses). The child’s clinical condition subsequently improved, as did the results of routine and radiological examinations. She became oxygen-independent after 15 days, and was discharged 20 days after beginning ciprofloxacin (i.e. 30 days after admission) with a negative M. pneumoniae PCR and no antibiotic therapy. No ciprofloxacin-related adverse event was observed during the following 3 months, at the end of which a chest CT scan appeared normal. DNA extracted from the respiratory samples using a Nuclisens EasyMAG automated extraction system (Biomerie´ux, Craponne, France) was pretested by means of real-time PCR targeting a conserved inter-repetitive region of the P1 gene of M. pneumoniae [8], and the M. pneumoniae-positive samples were characterised in known subtypes and variants by sequencing a variable part of the RepMP2/3 repetitive element of the P1 gene as recently described [9]. Briefly, the nested PCR procedure amplifies a part of RepMP2/3 that shows characteristic EUROPEAN RESPIRATORY JOURNAL
TABLE 1
Clinical and laboratory findings in a 6-yr-old girl with community-acquired pneumonia and clarithromycin-induced Mycoplasma pneumoniae macrolide resistance
Characteristic Axillary temperature# uC Respiratory rate
Day 0
Day 4
Day 10
Day 15
Day 25
Day 30
40.5
39.0
38.3
Apyrexia
Apyrexia
Apyrexia
60
55
52
45
25
24 98
breaths?min-1 Sp,O2" %
85
88
85
90
95
Heart rate beats?min-1
135
139
140
110
82
76
Blood pressure mmHg
90/60
93/61
88/58
91/62
95/65
90/60
Multiple ‘‘ground-
NE
‘‘Ground-glass’’
NE
Significantly
NE
Chest radiography
glass’’ infiltrates Chest CT scan
NE
opacifications Bilateral infiltrates,
improved
NE
NE
NE
NE
11860
11280
4900
6790
with ‘‘ground-glass’’ opacifications 10100
White blood cell count
NE
cells?mL-1 Neutrophils %
79
NE
82
84
41
60
CRP mg?dL-1
14.2
7.1
7.1
3.9
0.8
,0.3
ESR mm first hour
53
NE
48
NE
NE
11
Bacterial findings
M. pneumoniae DNA
M. pneumoniae DNA
M. pneumoniae DNA
M. pneumoniae DNA
M. pneumoniae DNA
Negative
in nasopharyngeal
in nasopharyngeal
in nasopharyngeal
in nasopharyngeal
swab
swab
swab and BAL
swab
Genotype Mutation of 23S rRNA Viral findings Antibiotic therapy
in nasopharyngeal swab
Variant 2a
Variant 2a
Variant 2a
Variant 2a
Variant 2a
NE
None
None
A2063G
A2063G
A2063G
NE Negative
Negative
Negative
Negative
Negative
Negative
Ampicillin/sulbactam
Meropenem and
Meropenem and
Meropenem and
Ciprofloxacin
and clarithromycin
teicoplanin and
teicoplanin and
teicoplanin and
clarithromycin
clarithromycin
ciprofloxacin
6
6
4
Supportive O2 therapy
8
End of ciprofloxacin
NR
NR
L?min-1 Sp,O2: arterial oxygen saturation measured by pulse oximetry; CT: computed tomography; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; rRNA: ribosomal RNA; BAL: bronchoalveolar lavage; NE: not evaluated; NR: not required. #: peak day value; ": in room air.
conserved differences in the sequences of the known subtypes and variants of M. pneumoniae; sequencing the PCR product allows the reliable classification of the detected strains. Mutations of the 23S rRNA gene of M. pneumoniae at positions relevant for most cases of macrolide resistance (2,063 and 2,064) were detected in a culture-independent manner as recently described [7]. Briefly, an aliquot of the product of the nested PCR amplifying a part of the 23S rRNA gene was used as a template for real-time PCR, and the putative mutations at positions 2,063/ 2,064, suggested by melting peak analysis were confirmed by means of sequence analyses, which also revealed the occurrence of mutations at position 2,617. Molecular subtyping showed a variant 2a in all of the investigated M. pneumoniae-positive samples, and an A-G transition at nucleotide 2,063 was found in the samples collected on days 10, 15 and 25, but not in those collected on day 0 and 4 (table 1).
in the 23S rRNA gene associated with in vitro resistance to macrolides. This is important because there has recently been a surge in macrolide resistance among clinical isolates of M. pneumoniae in various parts of the world. However, children in Europe are not usually involved and there is no report of resistance arising during the course of macrolide treatment.
This is the first case report showing the detection of macrolideresistance in M. pneumoniae not at admission but during treatment with a macrolide (clarithromycin). The child did not respond to i.v. clarithromycin administered at the usually recommended doses, but showed clinical resolution with ciprofloxacin. After 10 days of therapy (but not upon admission), it was found that the M. pneumoniae DNA had a mutation
Our case offers some insights into problems relating to the clinical significance of macrolide-resistant M. pneumoniae and the antibacterial treatment of the resulting infections. First of all, it supports the hypothesis that the emergence of macrolideresistant strains may also be related to drug administration as the mutation was only found after 10 days of clarithromycin treatment and the patient had no known predisposing factor related to the development of macrolide resistance (including immunosuppressive therapy). It is possible that this mechanism of resistance induction has been rarely reported because most previous studies have only considered samples obtained once during the course of the disease. An alternative intriguing explanation could be that the 23S rRNA mutation was already present in the M. pneumoniae population (at low levels) within the patient before the administration of antibiotics. The treatment of the patient with clarithromycin may have selected for outgrowth of those bacteria from the population that carried
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the 23S rRNA mutation. According to this last hypothesis, the PCR results may only represent the most prominent genotype of the M. pneumoniae population and could explain why only a ‘‘non-resistant’’ genotype was found at day 0 and 4, and only a ‘‘resistant genotype’’ was found at day 10.
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, and "Dresden University of Technology, Carl Gustav Carus Medical Faculty, Institute of Medical Microbiology and Hygiene, Dresden, Germany.
Moreover, our case confirms that the M. pneumoniae DNA with the A2,063G mutation can be associated with severe disease even when it infects otherwise healthy children. Although macrolide-resistant M. pneumoniae strains have been associated with prolonged fever in children [3], there are still no published data indicating that this resistant variant is inherently more pathogenic in any human host. Additionally, the appearance of macrolide resistance has been described in subtypes 1 and 2, which are characterised by sequence differences in all RepMP2/3, RepMP4 and RepMP5 repetitive elements [9]. Although the rate of variant 2a strains has increased in Europe [10], this is the first detection of macrolide resistance in this genotype.
Correspondence: S. Esposito, Dept of Maternal and Paediatric Sciences, Universita` degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Via Commenda 9, 20122 Milano, Italy. E-mail:
[email protected]
Finally, the optimal treatment for a serious infection caused by macrolide-resistant M. pneumoniae remains uncertain. Pharmacokinetics and in vitro data indicate that fluoroquinolones and tetracyclines are potential options, but both are contraindicated in children. We administered ciprofloxacin, which slowly led to a favourable clinical and microbiological response without any adverse events. This suggests that the potential benefits of this drug could be considered in the management of serious infections caused by macrolide-resistant M. pneumoniae. Another possibility is to verify whether high clarithromycin doses improve its antimicrobial efficacy and lead to a significantly greater improvement in the markers of disease severity without increasing the incidence or severity of adverse events. It has also been shown in a gnotobiotic mouse model mono-associated with macrolide-resistant M. pneumoniae that clarithromycin decreased the number of macrolide-sensitive and -resistant M. pneumoniae by modulating pulmonary inflammation [11]. In conclusion, our case shows that an A-G transition at position 2,063 of the 23S rRNA gene of M. pneumoniae can be detected during adequate treatment with clarithromycin and seems to be associated with treatment failure. It also highlights the fact that local surveillance using molecular methods may be important in determining the prevalence of macrolide resistance among M. pneumoniae strains. Furthermore, in the case of macrolide failure during the course of treatment, the presence of macrolide resistance needs to be assessed in order to optimise the antibiotic strategy rapidly. F. Cardinale*, M. Chironna#, R. Dumke", A. Binetti*, C. Daleno+, A. Sallustio#, A. Valzano+ and S. Esposito+ *Dept of Paediatric Allergy and Pulmonology, Giovanni XXIII Paediatric Hospital, University of Bari, #Hygiene Section, Dept of Biomedical Sciences and Human Oncology, Azienda Ospedaliero-Universitaria Policlinico, University of Bari, Bari, + Dept of Maternal and Paediatric Sciences, University of Milan,
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Support Statement: This letter was supported in part by a grant from the Italian Ministry of Health (Bando Giovani Ricercatori 2007). Statement of Interest: None declared.
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